The intracellular interplay between galectin-1 and FGF12 in the assembly of ribosome biogenesis complex

Galectins constitute a class of lectins that specifically interact with β-galactoside sugars in glycoconjugates and are implicated in diverse cellular processes, including transport, autophagy or signaling. Since most of the activity of galectins depends on their ability to bind sugar chains, galectins exert their functions mainly in the extracellular space or at the cell surface, which are microenvironments highly enriched in glycoconjugates. Galectins are also abundant inside cells, but their specific intracellular functions are largely unknown. Here we report that galectin-1, -3, -7 and -8 directly interact with the proteinaceous core of fibroblast growth factor 12 (FGF12) in the cytosol and in nucleus. We demonstrate that binding of galectin-1 to FGF12 in the cytosol blocks FGF12 secretion. Furthermore, we show that intracellular galectin-1 affects the assembly of FGF12-containing nuclear/nucleolar ribosome biogenesis complexes consisting of NOLC1 and TCOF1. Our data provide a new link between galectins and FGF proteins, revealing an unexpected glycosylation-independent intracellular interplay between these groups of proteins. Supplementary Information The online version contains supplementary material available at 10.1186/s12964-024-01558-1.


Background
Fibroblast growth factors (FGFs) and their receptors (FGFRs) constitute signaling hubs that regulate human cell and body homeostasis [1].The twenty-two FGF proteins are divided into seven subfamilies [2].One of these is the fibroblast growth factor homologous factors (FHFs): FGF11 (FHF3), FGF12 (FHF1), FGF13 (FHF2) and FGF14 (FHF4), whose members are widely expressed in various cell and tissue types, such as fibroblasts, cardiomyocytes, neurons and osteoclasts [3].FGF11-14 proteins do not have the ER-targeting signal peptide and were therefore for a long time considered solely intracellular proteins [3].However, recent studies have demonstrated that a small portion of the FGF11-14 pool is secreted in the unconventional manner, and when present outside cells, they can directly bind FGFRs, triggering anti-apoptotic signaling [4,5].
The vast majority of secreted FGFs and all FGFRs are N-glycosylated, and this modification affects their stability, adjusts the interaction network and modifies subcellular localization [27][28][29][30][31].We have recently demonstrated that the extracellular galectins, a family of multivalent lectins, decode information stored in N-glycan chains attached to FGFRs on the cell surface and secreted FGFs to fine-tune FGF/FGFR signaling [31][32][33][34][35][36][37].Galectins are also found inside the cell, where they interact with several proteins binding their proteinaceous cores, participating among the others in protein trafficking, signal transduction and apoptosis [38].In light of the significant role of galectins in N-glycan-dependent FGF/FGFR signaling and in the search for regulators of nuclear FGF12, we tested the interplay between intracellular FGF12 and selected members of the galectin family.

siRNA transfection
siRNA transfections were performed with Dharma-FECT Transfection Reagents (Horizon, Cambridge, UK) according to the manufacturer's instructions.Cells were transfected with 50 nM siRNA against galectin-1 or 50 nM non-targeting siRNA as a control.After 24 h, the transfection medium was replaced with the complete medium.Cells were incubated in a 5% CO 2 atmosphere at 37 °C for another 24 h.

Proximity ligation assay (PLA)
To analyze the interaction between studied proteins, Duolink ® In Situ Fluorescence Protocol was used (Sigma-Aldrich).U2OS-FGF12-GFP.myccells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton in PBS.Cells were then incubated with appropriate antibodies and treated according to the manufacturer's protocols.Cell nuclei were stained with NucBlue Live dye.Cytoplasm was stained with HCS Cell Mask Deep Red dye.

Fluorescence microscopy
Wide-field fluorescence microscopy was carried out using a Zeiss Axio Observer Z1 fluorescence microscope (Zeiss, Oberkochen, Germany) as described previously [21].At least three independent experiments were carried out and at least 50 cells were used for quantification per experiment.Confocal fluorescence microscopy measurements were carried out using Opera Phenix Plus High-Content Screening system (Perkin Elmer, Waltham, MA, USA).Fixed cells were

Analysis of galectin-1 impact on FGF12 secretion
After galecin-1 silencing, secretion analysis was performed in U2OS-FGF12-GFP.myccells at 42°C, as previously described [5].Media samples and cell lysates were analyzed by SDS-PAGE and western blotting with anti-FGF13 antibody, which is directed against peptide (AAAIASSLIRQKRQARE) present in FGF13 and FGF12.The amount of detected protein was quantified using ImageLab Software.Three independent experiments were quantified.
Using proximity ligation assay (PLA), we tested whether intracellular galectins are capable of interacting with FGF12.As demonstrated in Fig. 1B, we detected significant PLA signals for all tested galectins and FGF12, with the strongest signal measured for the galectin-1/ FGF12 pair.Importantly, for all galectins studied, the PLA signal was predominantly localized to the nucleus (Fig. 1B).In the case of galectin-1, we also observed strong PLA signals in the cytosol (Fig. 1B).These data indicate that galectin-1, -3, -7 and -8 form intracellular complexes with FGF12, and these complexes are mainly present in the nucleus.

Galectins directly bind the proteinaceous core of FGF12
Since PLA experiments cannot accurately distinguish a direct interaction from an indirect one, we produced recombinant galectin-1, -3, -7 and -8 and FGF12 in a bacterial expression system and used the resulting proteins to measure intermolecular interactions using surface plasmon resonance (SPR).SPR experiments revealed specific and direct interactions between all galectins tested and FGF12 (Fig. 2).The measured affinities (K D ) of galectins for FGF12 were in the micromolar (galectin -1, -3 and -7) or submicromolar range (galectin -8) (Fig. 2).Since in SPR assays we used recombinant galectins and FGF12 of bacterial origin incapable of glycosylation, our data suggest that the tested galectins interact directly with the proteinaceous core of FGF12.Interestingly, the affinities of galectins for FGF12 are in a similar range to the values obtained for galectin/Nglycosylated FGFs pairs [32].All these data suggest that galectin-1, -3, -7 and -8 directly interact with FGF12.

Galectin-1 is a negative regulator of FGF12 secretion
In further studies, we decided to focus on the intracellular interplay between galectin-1 and FGF12, since among the galectins tested, we were able to effectively knock-down only galectin-1 in U2OS-FGF12-GFP.mycFig. 3 Role of galectin-1 in regulation of FGF12 secretion A Galectin-1 knock-down in U2OS-FGF12-GFP.myccells.Western blotting analysis of cell lysates of U2OS-FGF12-GFP.myccells treated with siRNA against galectin-1 and scramble siRNA as a control.The amount of detected galectin-1 was quantified using densitometry measurements with ImageLab Software (right panel).Mean values from three independent experiments ± SD are shown.Statistical analyses were performed with Student's t-test (*p < 0.05; **p < 0.005 and ***p < 0.001).B Impact of galectin-1 on FGF12 secretion.U20S and U2OS-FGF12-GFP.myccells after galectin-1 knock-down were serum-starved at 37°C for 24 h and, after medium exchange, incubated at 42°C for 2 h.The medium from above the cells was collected, centrifuged and incubated with anti-myc-tag magnetic beads.50% of eluted samples were loaded onto SDS-PAGE gels and analyzed by western blotting.FGF12 protein was detected with anti-FGF13 antibody, recognizing all proteins belonging to FHF family.The amount of detected protein was quantified using densitometry measurements in ImageLab Software cells (Fig. 3A).We have recently reported that FGF12-GFP.myc is secreted by U2OS cells [5].The efficiency of unconventional FGF12 secretion was increased by elevated but the molecular mechanism of FGF12 release is still unknown [5].To study whether the intracellular interaction between galectin-1 and FGF12 might affect FGF12 secretion, we analyzed the amounts of FGF12-GFP.mycfound in the cell culture media upon galectin-1 knock-down with siRNA.As shown in Fig. 3B, depletion of U2OS-FGF12-GFP.myccells of galectin-1 resulted in significantly level of extracellular FGF12-GFP.myc.Based on these findings, we hypothesize that highly abundant galectin-1 forms a stable complex with FGF12 in the cytosol and nucleus, which impedes FGF12 translocation through the plasma membrane.

Galectin-1 constitutes a novel modulator of the FGF12/ NOLC1/TCOF1 complex
Previous studies have shown that nucleolar FGF12 interacts with NOLC1 and TCOF1 and that silencing of FGF12 results in a strongly reduced interaction between NOLC1 and TCOF1 [21].These data imply that FGF12 may act as a molecular bridge physically linking NOLC1 to TCOF1.Using PLA in conjunction with the high content quantitative confocal microscopy, we studied the involvement of galectin-1 in the interaction of FGF12 with NOLC1 and TCOF1 [21].To this end, we downregulated galectin-1 with siRNA and observed a significantly reduced interaction between FGF12 and NOLC1, and between FGF12 and TCOF1 (Fig. 4A and B).Interestingly, silencing of galectin-1 strongly enhanced the binding of NOLC1 to TCOF1 (Fig. 4C).These data implicate that galectin-1 directly binds FGF12 and promotes the interaction between FGF12 and NOLC1, and FGF12 and TCOF1.On the other hand, galectin-1 negatively regulates complex formation between NOLC1 and TCOF1, which is facilitated by FGF12.

Conclusions
In this work, we have provided a novel link between galectins and FGF/FGFR.In addition to the previously described N-glycosylation-dependent action of Fig. 5 Hypothetical model of interplay between galectin-1, FGF12, NOLC1, TCOF1.Galectin-1 directly interacts with FGF12 in the cytosol and nucleus.Direct binding between these proteins inhibits the secretion of FGF12 (A).The nuclear function of FGF12-galectin-1 complexes remains unknown (B), but galectin-1 promotes the interaction of FGF12 with NOLC1 (C) and TCOF1 (D) and downregulates the binding of NOLC1 to TCOF1 (E) extracellular galectins on the glycosylated FGFRs and FGFs, our data suggest the presence of an intracellular interplay between galectins and FGF12 that is based on the direct interaction between proteinaceous cores of these protetins.Based on these data, we hypothesize that there is a dynamic interplay between galectin-1, FGF12, NOLC1 and TCOF1 that occurs in several subcellular compartments (Fig. 5).Galectin-1 directly interacts with the proteinaceous core of FGF12 to form galectin-1/ FGF12 complexes in the cytosol and nucleus.By binding FGF12, galectin-1 captures FGF12 inside the cell and thus impedes the unconventional secretion of FGF12 (Fig. 5A).In the nucleus, galectin-1 affects the interplay between FGF12, NOLC1 and TCOF1.Galectin-1 promotes the assembly of FGF12/NOLC1 and FGF12/ TCOF1 complexes (Fig. 5B and C) and inhibits the interaction between NOLC1 and TCOF1 (Fig. 5E).Further studies are needed to decipher the involvement of interplay between galectin-1, FGF12, NOLC1 and TCOF1 in nuclear homeostasis and function.

Fig. 4
Fig.3Role of galectin-1 in regulation of FGF12 secretion A Galectin-1 knock-down in U2OS-FGF12-GFP.myccells.Western blotting analysis of cell lysates of U2OS-FGF12-GFP.myccells treated with siRNA against galectin-1 and scramble siRNA as a control.The amount of detected galectin-1 was quantified using densitometry measurements with ImageLab Software (right panel).Mean values from three independent experiments ± SD are shown.Statistical analyses were performed with Student's t-test (*p < 0.05; **p < 0.005 and ***p < 0.001).B Impact of galectin-1 on FGF12 secretion.U20S and U2OS-FGF12-GFP.myccells after galectin-1 knock-down were serum-starved at 37°C for 24 h and, after medium exchange, incubated at 42°C for 2 h.The medium from above the cells was collected, centrifuged and incubated with anti-myc-tag magnetic beads.50% of eluted samples were loaded onto SDS-PAGE gels and analyzed by western blotting.FGF12 protein was detected with anti-FGF13 antibody, recognizing all proteins belonging to FHF family.The amount of detected protein was quantified using densitometry measurements in ImageLab Software (right panel).Mean values ± SD from three independent experiments are shown.Statistical analyses were performed with Student's t-test (*p < 0.05; **p < 0.005 and ***p < 0.001)